New mAbs facilitate quantification of secreted equine TNF-α and flow cytometric analysis in monocytes and T cells.

This research article describes a new means to measure the levels of Tumor Necrosis Factor-a (TNF-α), an inflammatory marker, in horses. The researchers developed two new monoclonal antibodies (mAbs) for this purpose and applied them in lab testing scenarios.

Background

• Monoclonal antibodies are engineered antibodies that can specifically bind to a certain protein. In this study, they were designed to target and detect equine TNF-α, an important biomarker in inflammation.
• Equine TNF-α quantification through existing tools had limitations, which this study aimed to address with two newly generated mAbs (clones 48 and 292).

Methods

• The research team evaluated the specificity of the two mAbs to TNF-α and confirmed their ability to detect native TNF-α in stimulated equine Peripheral Blood Mononuclear Cells (PBMC).
• The mAbs were then employed in a fluorescent bead-based assay, a technique that utilizes tiny fluorescent beads to detect specific proteins, for quantification of equine TNF-α.
• mAb 48, in particular, was used for a detailed analysis of TNF-α production in PBMC by intracellular staining and flow cytometry, a technique used to measure physical and chemical characteristics of cells or particles.

Results

• The TNF-α assay, which uses mAbs to quantify TNF-α levels, displayed a wide range of sensitivity, from as low as 12 pg/mL up to 38.4 ng/mL.
• Under lab conditions, it was found that TNF-α was produced by CD14 monocytes once they were stimulated with Lipopolysaccharides (LPS), a common bacterial component that elicits a strong immune response.
• It was also found that TNF-α was expressed by both monocytes (a type of white blood cell) and lymphocytes (another type of white blood cell) after they were stimulated with Phorbol 12-myristate 13-acetate (PMA) and ionomycin, two chemical compounds that trigger cellular responses.
• Among the lymphocytes, CD4 T cells primarily expressed TNF-α. However, TNF-α expression was also observed in CD8 T cells and other lymphocyte types.

Conclusion

• This study introduced two new mAbs that effectively quantify and analyze TNF-α during inflammatory responses in horses.
• This technique could enhance our understanding of immune responses and inflammation in horses, thereby promoting the development of therapeutic strategies for equine inflammatory diseases.